Diabetes is a disease that affects hundreds of millions of people worldwide. Central to this disease is the insulin-producing beta cell, which is found within islets of Langerhans in the pancreas. These cells are damaged or destroyed in diabetic patients. A virtually-unlimited number of beta cells can be generated in vitro from human embryonic stem cells and human induced pluripotent stem cells, potentially from patients. While current approaches generate beta-like cells from stem cells in vitro that resemble bone fide beta cells from islets in vivo, these cells still lack several hallmarks of full maturity, including gene expression and mounting a proper insulin secretion response to glucose. The focus of this research proposal is to understand how endothelial cells and extracellular matrix proteins influence differentiation to and maturation of human beta-like cells from stem cells. These components are found in the islet but are absent from current stem cell differentiation protocols that produce beta-like cells. The proposed studies leverage a newly-developed three- dimensional hydrogel culture system to assemble stem cell derivatives into islet organoids to interrogate the relationship between beta-like cells and these islet microenvironmental components. Understanding this relationship will provide fully mature beta-like cells for the study and treatment of diabetes.